Mechanism of action of cutinase: chemical modification of the catalytic triad characteristic for serine hydrolases.
Cutinase from Fusarium solani f. sp. pisi was inhibited by diisopropyl fluorophosphate and phenylboronic acid, indicating the involvement of an active serine residue in enzyme catalysis. Quantitation of the number of phosphorylated serines showed that modification of one residue resulted in complete loss of enzyme activity. One essential histidine residue was modified with diethyl pyrocarbonate. This residue was buried in native cutinase and became accessible to chemical modification only after unfolding of the enzyme by sodium dodecyl sulfate. The modification of carboxyl groups with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the absence of sodium dodecyl sulfate did not result in inactivation of the enzyme; however, such modifications in the presence of sodium dodecyl sulfate resulted in complete loss of enzyme activity. The number of residues modified was determined by incorporation of [14C]glycine ethyl ester. Modification of cutinase in the absence of sodium dodecyl sulfate and subsequent unfolding of the enzyme with detergent in the presence of radioactive glycine ester showed that one buried carboxyl group per molecule of cutinase resulted in complete inactivation of the enzyme. Three additional peripheral carboxyl groups were modified in the presence of sodium dodecyl sulfate. Carbethoxylation of the essential histidine and subsequent incubation with the esterase substrate p-nitrophenyl [1-14C]acetate revealed that carbethoxycutinase was about 10(5) times less active than the untreated enzyme. The acyl-enzyme intermediate was stabilized under these conditions and was isolated by gel permeation chromatography. The results of the present chemical modification study indicate that catalysis by cutinase involves the catalytic triad and an acyl-enzyme intermediate, both characteristic for serine proteases.
D001665:Binding Sites; D002265:Carboxylic Ester Hydrolases / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D005670:Fusarium / Q000201:enzymology*; D005779:Immunodiffusion; D007700:Kinetics; D011485:Protein Binding; D012697:Serine Endopeptidases
D003432:Cross-Linking Reagents; D002265:Carboxylic Ester Hydrolases; C019952:cutinase; D010450:Endopeptidases; D012697:Serine Endopeptidases; D004047:Diethyl Pyrocarbonate; D005022:Ethyldimethylaminopropyl Carbodiimide